Biofabrication of a Fully Biological Gas Exchange Membrane for a Tissue-Based Lung
With less than 2,800 lung transplants per year in the U.S., long-term respiratory support is needed for the 15 million patients with chronic lung diseases. Extracorporeal membrane oxygenation (ECMO) can supplement patients’ gas exchange for months, but blood contact with ECMO oxygenators’ synthetic materials triggers clot formation. Systemic anticoagulants are used but must be limited to mitigate the risk of bleeding complications, and thus, the oxygenator clots, fails, and must be replaced every 1-4 weeks (Chapter 1). An endothelial cell coating like a blood vessel’s lining would reduce clotting, but long-term attachment of endothelial cells under shear stress has not been achieved on artificial materials (Chapter 2). The purpose of this dissertation is to tissue engineer a gas exchange membrane as an initial step toward creating the first, 100% biological, de novo, biofabricated lung. Chapter 3 provides a design overview of the macro-scale lung and the micro-scale tissue and then establishes their functional metrics.
The first of two experimental chapters, Chapter 4 addresses the need for thin but strong biological membranes. Methods are described for casting and dehydrating collagen I to achieve a rehydrated thickness of 18.8 ± 3.6 µm standard deviation and concentration of approximately 300 mg/mL. The membranes retained plasma while suspended in air and withstood ≥120 mmHg of liquid pressure. The collagen I adhered and supported a confluent vascular endothelium and lung epithelium in air-liquid culture, creating a fully biological membrane (<50 µm thick) that mimics the layers of the native alveolar capillary barrier. Live/dead cell stains revealed sufficient water permeability and nutrient transport to support at least one layer of epithelial cells. Large molecule permeability results indicated epithelial cells, not just endothelial cells, will be necessary for barrier function between blood and gas spaces. In particular, the epithelial cell layer will need to be exchanged with a cell type with tighter junctions to bring albumin permeability closer to in vivo levels. A custom bioreactor was also utilized to quantify gas transfer across the collagen I (~20 µm), relative to a polydimethylsiloxane (PDMS) elastomer (~51 µm). Studies found the collagen I to be less O2 permeable than the PDMS (p < 0.01). This result, in combination with theoretical diffusivity constants, suggests the collagen I membrane at this concentration should be relatively three to four-fold thinner (~14 µm) than the PDMS elastomer to match its performance. To decrease thickness further, low levels of chemical cross-linkers could be used to strengthen the collagen.
Chapter 5 then demonstrated the feasibility of forming perfusable, thin-walled, parallel plate channels made entirely out of collagen I. The collagen was cast around wax, dehydrated, and perfused with HistoClear II for wax removal. This provided channels with 37.7 ± 5.6 µm walls and four cm2 of fully biological area for gas exchange. As a step towards decreased wall thickness, channels were then made with a 16.8 ± 3.4 µm wall without chemical crosslinking. Lastly, a cell-safe bioreactor was designed, constructed, and validated that incorporates the cast conduits in an air-tight manner for use as a gas exchange test system. In summary, significant progress was made constructing a first generation, fully biological, gas exchange membrane. Future work should improve upon this membrane by using thinner (≤15 µm), lightly cross-linked, collagen I membranes and channels that support induced pluripotent stem cell-derived endothelial and epithelial cells.
- Biomedical Engineering
- Doctor of Philosophy (PhD)